Sample preconcentration in capillary electrophoresis is a powerful analytical technique suitable for the analysis of biological molecules like amino acids, peptides, proteins, nucleic acids, nucleotides, etc. As a liquid-phase separation technique, capillary electrophoresis (CE) possesses a number attractive features including high efficiency, short analysis times, small sample sizes, etc. However, with UV/vis detection, which is the most common detection scheme in CE, the concentration sensitivity of the technique is usually poor. Sample preconcentration is especially important for trace analyses. Studies have been done to increase the sample concentration sensibility in CE. Among these studies, on-line sample preconcentration methods account for a large part. Stacking is the major technique which is widely used for sample concentration in capillary zone electrophoresis (CZE). Sweeping is another important sample concentration method in CE.
CE is a type of electrophoresis, and involves resolving components in a mixture within a capillary to which an electric field is applied. The capillary used to conduct electrophoresis is filled with an electrolyte and a sample introduced into one end of the capillary using various methods such as hydrodynamic pressure, electro-osmotically-induced flow, and electrokinetic transport. The ends of the capillary are then placed in contact with an anode solution and a cathode solution and a voltage applied across the capillary. Positively charged ions are attracted towards the cathode, whereas negatively charged ions are attracted to the anode. Species with the highest mobility travel the fastest through the capillary matrix. However, the order of elution of each species, and even from which end of the capillary a species elutes, depends on its apparent mobility. Apparent mobility is the sum of a species electrophoretic mobility in the electrophoretic matrix and the mobility of the electrophoretic matrix itself relative to the capillary. The electrophoretic matrix may be mobilized by hydrodynamic pressure gradients across the capillary or by electro-osmotically-induced flow (electro-osmotic flow).
Capillary electrophoresis (CE) is a highly efficient separation technique, and possesses a number of advantageous features including high overall separation efficiency, short analysis times, and small amounts of reagents and/or samples required. CE also provides a biocompatible separation environment that is especially suitable for biological molecules including proteins, nucleic acids, peptides, nucleotides, amino acids etc. CE separations are often performed using an on-column detection mode to prevent loss of separation efficiency due to extra column band broadening that usually takes place if an off-column detection cell is used. However, in UV detection (which is the most commonly used detection technique in CE), due to the short path length (equal to the inner diameter of the column), on-column UV detection is characterized by low concentration sensitivity. A series of studies have been undertaken over the past few decades to increase the concentration sensibility in CE. Approaches used to address this problem can be divided into three categories: (a) sample preconcentration strategies, (b) alternative capillary geometry and improved optical design, and (c) alternative detection modes. The first technique, commonly called on-line sample preconcentration, is especially attractive since it involves no additional modification of the commercially available standard CE instrument, and it can be easily accomplished by carefully controlling the operation conditions on a standard CE instrument.
Stacking is one of the most widely used techniques for sample preconcentration in capillary zone electrophoresis (CZE). In “field amplified sample stacking”, the velocity of analyte ions is changed by using discontinuous buffers. When a high voltage is applied, a higher electric field is developed in the dilute sample plug than in the more concentrated running buffer because of the higher resistivity of the sample zone. The analyte ions then stack at the boundary between the sample plug and the running buffer, forming a narrow stacked zone. Discontinuous buffers can be prepared simply by addition of salts into buffer, or dissolving the sample in a low ionic strength buffer, or by adjusting their pH values. An enhancement factor of more than 100 is achieved for protein samples. For zwitterionic solutes like amino acids, peptides and proteins, discontinuity in the running buffer pH could be applied to achieve preconcentration and focusing. On-line focusing of flavin derivatives using dynamic pH junction in CE is reported to achieve a more than 1200-fold improvement in sensitivity relative to standard injection method in CE. When a large volume of sample is introduced into the separation column for stacking, the solute zone is as wide as the length of the sample plug. Several techniques have been developed to achieve a narrow stacked sample band for further analysis. Methods for stacking from a very large sample volume have been developed. First, a large volume of sample prepared in a dilute buffer is introduced into the column. A negative voltage is then applied at the capillary ends to obtain EOF directed towards the capillary inlet. Under these conditions, the sample matrix is gradually pushed out of the capillary by EOF and the anions stacked at the boundary between sample solution and the background electrolyte (BGE). The resulting sample zone is narrow, and high-efficiency separation capabilities in CE are preserved. This method is also used to determine some quaternary ammonium herbicides in spiked drinking water.
Another focusing technique in CE is capillary isoelectric focusing (CIEF). This method utilizes the differences in the isoelectric points (pls) of analytes. The separation capillary is filled with a solution of ampholytes. If an electric field is applied across such a capillary, a pH gradient is generated along its length, and the zwitterionic analytes in the subsequently injected sample begin to migrate through the capillary under applied field. Each of the analytes will lose its net charge when it reaches the location in capillary where the pH of the ampholyte equals to the pl of the analyte. In the absence of EOF, focused discrete neutral analyte zones line up inside the capillary at locations corresponding to their pl values. CIEF is widely used for the analysis of analytes having different pl values. For example, an enhancement factor of 500 was achieved for polypeptide mixtures resulting from digestion of proteins, even though the components of the resulting mixture had very small differences in isoelectric points (Δpl˜0.01).
Capillary isotachophoresis (CITP) can also be employed for sample stacking. CITP is accomplished in a capillary by injecting the sample between two discrete buffer plugs: a leading buffer with a higher mobility ion, and a terminating buffer having a lower mobility ion, than the charged analytes. When an electric field with the constant current is applied, the ions inside the sample are distributed into narrow and concentrated zones between leading and terminating buffer based on the differences in their mobilities. CITP has been used to on-line preconcentrate and separate inorganic, organic, and biomolecules.
With the development in microchip-based capillary electrophoresis, sample stacking techniques have also been used in the microfluidic CE devices. A few orders of magnitude in sample enrichment have been obtained by stacking in microchip-based CE.
The concept of sample sweeping has also been introduced. Sample sweeping is accomplished in micellar electrokinetic chromatography (MEKC), in which micelles act as a pseudo-stationary phase and “sweep” the analytes from the long injected sample plug and converts it into narrow zone(s), thereby preconcentrating the analyte(s) from the wide band of originally injected dilute sample. Sweeping makes it possible to preconcentrate neutral analytes. A million-fold sensitivity increase has been reported with a combination of stacking and sweeping effects.
A different strategy to preconcentrate neutral analytes in MEKC includes, contrary to preparing sample in a dilute, low conductivity electrolyte commonly used in sample stacking process uses a high-conductivity sample matrix, which enables the micelles to be focused before they enter the sample zone. Their method also solves the problem for the preconcentration of samples having high salt content, which is frequently met in real life situation.
The stacking of ionizable analytes in high salt sample matrix by means of transient moving chemical reaction boundary method (tMCRBM) has also been reported. Sample got stacked in the tMCRBM generated between two phases (a weak acid of the running buffer and a weak base of sample matrix). The mechanism is dependent on the zwitterionic properties of the analytes that change their net charges based on the pH. The high salt concentration in sample matrix slows down the migration velocities of analytes producing a narrow, stacked zone.
Solid-phase extraction (SPE) is another important sample preconcentration strategy. With this method, multiple column volumes of sample can be injected since the analytes are adsorbed on the stationary phase. SPE can be coupled to capillary electrophoresis system, where the preconcentrated samples get separated on the CE column. The extraction also could be accomplished by ion-exchange procedure.
What is needed is an improved method for increasing the sample concentration sensibility in CE, which should yield trace detections of analyte by UV detector, in preconcentrated samples.